Costamere Protein Expression and Tissue Composition of Rotator Cuff Muscle After Tendon Release in Sheep

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Year: 2018

Costamere protein expression and tissue composition of rotator cuff muscle after tendon release in sheep

Ruoss, Severin ; Möhl, Christoph B ; Benn, Mario C ; von Rechenberg, Brigitte ; Wieser, Karl ; Meyer, Dominik C ; Gerber, Christian ; Flück, Martin

Abstract: Previous studies suggested that degradation of contractile tissue requires cleavage of the costamere, a structural protein complex that holds sarcomeres in place. This study examined if costamere turnover is affected by a rotator cuff tear in a previously established ovine model. We found theactivity of focal adhesion kinase (FAK), a main regulator of costamere turnover, was unchanged at 2 weeks but decreased by 27% 16 weeks after surgical release of the infraspinatus tendon. This was accompanied by cleavage of the costamere protein talin into a 190 kDa fragment while full length talin remained unchanged. At 2 weeks after tendon release, muscle volume decreased by 17 cm from an initial 185 cm(3) , the fatty tissue volume was halved, and the contractile tissue volume remained unchanged. After 16 weeks, the muscle volume decreased by 36 cm(3) , contractile tissue was quantitatively lost, and the fat content increased by 184%. Nandrolone administration mitigated the loss of contractile tissue by 26% and prevented fat accumulation, alterations in FAK activity, and talin cleavage. Taken together, these findings imply that muscle remodeling after tendon release occurs in two stages. The early decreaseof muscle volume is associated with reduction of fat; while, the second stage is characterized by substantial loss of contractile tissue accompanied by massive fat accumulation. Regulation of costamere turnover is associated with the loss of contractile tissue and seems to be impacted by nandrolone treatment. Clini- cally, the costamere may represent a potential intervention target to mitigate muscle loss after a rotator cuff tear. © 2017 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc.on behalf of the Orthopaedic Research Society. J Orthop Res.

DOI: https://doi.org/10.1002/jor.23624

Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-141096 Journal Article Published Version

Originally published at: Ruoss, Severin; Möhl, Christoph B; Benn, Mario C; von Rechenberg, Brigitte; Wieser, Karl; Meyer, Do- minik C; Gerber, Christian; Flück, Martin (2018). Costamere protein expression and tissue composition of rotator cuff muscle after tendon release in sheep. Journal of Orthopaedic Research, 36(1):272-281. DOI: https://doi.org/10.1002/jor.23624 Costamere Protein Expression and Tissue Composition of Rotator Cuff Muscle After Tendon Release in Sheep

Severin Ruoss ,1 Christoph B. Mohl,€ 1 Mario C. Benn,2 Brigitte von Rechenberg,2 Karl Wieser,3 Dominik C. Meyer,3 Christian Gerber,3 Martin Fluck€ 1

1Laboratory for Muscle Plasticity, Balgrist Campus, University of Zurich, Zurich, Switzerland, 2Vetsuisse Faculty, University of Zurich, Zurich, Switzerland, 3Department of Orthopaedics, Balgrist University Hospital, University of Zurich, Zurich, Switzerland Received 25 November 2016; accepted 24 May 2017 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.23624

ABSTRACT: Previous studies suggested that degradation of contractile tissue requires cleavage of the costamere, a structural protein complex that holds sarcomeres in place. This study examined if costamere turnover is affected by a rotator cuff tear in a previously established ovine model. We found the activity of focal adhesion kinase (FAK), a main regulator of costamere turnover, was unchanged at 2 weeks but decreased by 27% 16 weeks after surgical release of the infraspinatus tendon. This was accompanied by cleavage of the costamere protein talin into a 190 kDa fragment while full length talin remained unchanged. At 2 weeks after tendon release, muscle volume decreased by 17 cm3 from an initial 185 cm3, the fatty tissue volume was halved, and the contractile tissue volume remained unchanged. After 16 weeks, the muscle volume decreased by 36 cm3, contractile tissue was quantitatively lost, and the fat content increased by 184%. Nandrolone administration mitigated the loss of contractile tissue by 26% and prevented fat accumulation, alterations in FAK activity, and talin cleavage. Taken together, these ﬁndings imply that muscle remodeling after tendon release occurs in two stages. The early decrease of muscle volume is associated with reduction of fat; while, the second stage is characterized by substantial loss of contractile tissue accompanied by massive fat accumulation. Regulation of costamere turnover is associated with the loss of contractile tissue and seems to be impacted by nandrolone treatment. Clinically, the costamere may represent a potential intervention target to mitigate muscle loss after a rotator cuff tear. ß 2017 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res

Keywords: rotator cuff tear; fatty infiltration; atrophy; costamere; tenotomy

Tears of rotator cuff (RC) tendons are frequent and degraded,10 as they are held in place by the costamere, a affect a large portion of the elderly population.1,2 structural protein complex that links the Z-line to the These tears lead to retraction, fat accumulation, and extracellular matrix.11 Therefore, longitudinal atrophy atrophy of the musculotendinous unit.3 To reestablish first requires disruption of the structural anchors (i.e., normal shoulder function, RC tendon tears are often the costameres) by specific Ca2þ-activated proteases12 to surgically repaired, and the degree of muscle atrophy release the respective sarcomeres. In fact, Z-line stream- and fat accumulation correlates with the probability of ing is observed after tendon release in the rat13 and after repair failure.4,5 Until recently, these tendon tear- spontaneous tendon tears in various human muscles.14 induced changes were considered irreversible.3,6 Thus, The main regulator of costamere turnover is focal it is important to characterize these alterations to find adhesion kinase (FAK), an integrin-associated phos- solutions for their prevention and treatment and allow photransfer kinase that is activated by phosphoryla- a successful recovery from a RC tear. tion on its tyrosine 397 site (pY397). It is Tendon release induces a broad range of adaptations mechanosensitive and reacts to altered loading of the in the muscle, with reduced muscle length most fre- muscle.15 Changes in protein concentrations of FAK quently described.7 Regulation of muscle length occurs and FAK-pY397 after reduced muscle loading, for by adding (longitudinal hypertrophy) or removing (longi- example, unloading of human m. vastus lateralis16 or tudinal atrophy) contractile material (i.e., sarcomeres) at tendon release of the rat m. soleus17 are well de- theendsofmyofibrils8 and it has been shown that scribed. However, it is currently unknown whether tendon release leads to a reduced number of sarcomeres FAK, FAK-pY397, and concomitant alterations in in series.9 Sarcomeres are tightly incorporated in costamere turnover are affected by RC tears. a myofibril and cannot simply be removed and/or Apart from early surgical repair, another treatment option, which has been described in sheep6 and rabbits,18 is weekly administration of the anabolic This is an open access article under the terms of the Creative steroid nandrolone decanoate, which prevented fat Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the accumulation when treatment was started with the original work is properly cited, the use is non-commercial and no onset of the tear. To our knowledge, the action of modifications or adaptations are made. anabolic steroids on the costamere is not yet de- Conflicts of interest: The authors declare that they have no scribed after tendon release. Nevertheless, nandro- competing interests. 2þ Grant sponsor: RESORTHO; Grant sponsor: Schweizerischer lone decreased cytosolic Ca in the mouse m. soleus Nationalfonds zur Forderung€ der Wissenschaftlichen Forschung; after hindlimb unloading.19 This may result in lower Grant number: 149786. Ca2þ-activated protease activity and thereby de- Correspondence to: Martin Fluck€ (T: þ41-44-510-73-50; E-mail: mfl[email protected]) creased costamere cleavage, which potentially explains protection from muscle loss.10,12,20 In the # 2017 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. ovine model of RC tear, the reduction of total muscle

JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2017 1 2 RUOSS ET AL. volume after tendon release did not differ with or Single Step Repair without nandrolone administration; although, fat The details of this procedure, which involves removal of the accumulation was prevented by nandrolone.6 This silicon wrap and reattachment of the greater tuberosity to its 6 model has been used to replicate chronic tears (e.g., original site, have been previously described. at 16–40 weeks)6,21,22 with high intramuscular fat content (around 50%; Goutallier stage 323). However, Sampling of Muscle Tissue Approximately 40 mg of tissue (0.02% of average PRE muscle early alterations of muscle volume and composition volume) was collected intraoperatively using a Bergstroem (i.e., representing an acute tear after 2 weeks) and needle with a 5 mm diameter (Dixons Surgical Instruments the impact of nandrolone on the costamere complex LTD, Wickford, UK). If necessary, samples were rapidly have not been investigated yet. cleaned of blood and immediately frozen in liquid nitrogen Therefore, the aim of the current study was (i) to cooled isopentane and stored in 2 ml cryotubes at À80˚C. define RC muscle volume and composition after tendon release in sheep and (ii) to describe alterations of Assessment of Tissue Composition on Biopsy Cross-Section costamere turnover with and without weekly doses of To assess the tissue composition of the biopsy specimens, 2 nandrolone. We specifically hypothesized that muscle consecutive sections (average section area: 6.54 mm ) were either stained with oil red O to detect fatty tissue (Fig. 1, remodeling starts early after a RC tear and is already 24 detectable after 2 weeks. Additionally, we hypothe- right), or using the Goldner trichrome technique to stain muscle fibers and connective tissue (Fig. 1, left). For the oil sized that changes in muscle volume and composition red O staining, sections were fixed for 10 min in 4% are accompanied by decreased FAK phosphorylation paraformaldehyde. After washing, sections were incubated and increased costamere turnover, assessed by the for 5 min in 60% isopropanol and then stained for 10 min in associated protein levels of FAK, talin, and vinculin. the oil red O working solution (40% of 5 g/L oil red O [#0684- Furthermore, we hypothesized that nandrolone admin- 100G, VWR, Radnor, PA] in isopropanol; 60% ddH2O). Then, istration not only prevents fat accumulation, but also sections were again incubated in 60% isopropanol, washed, reduces the loss of contractile tissue and mitigates counterstained in hematoxylin, and then rinsed in costamere cleavage. ddH2O before coverslips were mounted. The total area was determined on the oil red O stained METHODS section with ImageJ (v1.48v, National Institutes of Health, USA) using the “analyze particles” tool. Afterward, red Experimental Design This experiment was performed according to the Swiss law of stained areas (i.e., fatty tissue) were visually isolated, animal welfare (TSchG455) and approved by the Veterinary quantified with the same tool, and related to the total area. Office of the Canton of Zurich (No. 72/2013). The tendon of The area corresponding to contractile tissue was determined the m. infraspinatus was surgically released in 18 female from the red-colored muscle fibers on the Goldner stained Swiss Alpine sheep to simulate a RC tendon tear. Follow-up sections. Then, fatty tissue and contractile tissue were ¼ À measurements were performed either 2 weeks (TR2 group; related to the total area. The total area ( 100%) (fatty þ n ¼ 6; [mean Æ SD] age: 16.6 Æ 0.0 months; weight: tissue contractile tissue) was named “other types of tissue” 59.7 Æ 2.5 kg) or 16 weeks after tendon release (TR16 group; and covered all extramyocellular tissue apart from fat. n ¼ 6; age: 23.2 Æ 1.2 months; weight: 45.3 Æ 4.8 kg). In a third group, sheep underwent the same procedure as in the Assessment of Muscle Volume and Composition TR16 group, but also received weekly doses of 150 mg To determine muscle volume, MRI scans of both shoulders nandrolone decanoate in the m. gluteus maximus (TR16 þ were performed immediately after surgery with the sheep NAN group; n ¼ 6, age: 23.8 Æ 1.2 months; weight: still under general anesthesia. Scanning and readout 46.7 Æ 2.4 kg). Prior to tendon release (PRE) and 2 (2 W) or analysis were performed as described previously.6 The 16 weeks (16 W) after tendon release, alterations in muscle approximate volumes of the different tissue types in the volume were assessed by magnetic resonance imaging (MRI). total muscle were calculated by splitting the total volume Furthermore, at 16 W, the tendon was repaired in the TR16 according to the percentages of fatty, contractile, and other and TR16 þ NAN groups and measurements were performed tissue types that were assessed histologically in the biopsy again 6 weeks later (REP þ 6 W) to describe the effects of specimen. repair. Biopsy samples were collected from the m. infraspinatus to determine the tissue composition and costamere Immunoprecipitation of FAK-pY397 protein levels at the time points: PRE, 2 W, 16 W, and Muscle homogenates were prepared from cryosections of REP þ 6 W. The contralateral shoulder served as the control muscle biopsies as previously described.25 The protein (CC). Sheep were killed after harvesting the last biopsy, homogenate (250 mg) was brought to a total volume of 750 ml which was at 2 W for the TR2 group and at REP þ 6 W for with RIPA buffer. This was mixed for 30 min at 4˚C under the TR16 and TR16 þ NAN groups. steady rotation (20 rpm) and then centrifuged. Without touching the pellet, 700 ml of the homogenate was moved to a Surgical Tendon Release new tube and the centrifugation step was repeated. Then, Surgery was performed as described previously.6 In brief, the 650 ml of the homogenate was moved to a new tube and tendon of the m. infraspinatus was released via osteotomy of incubated over night with 200 mg protein A-sepharose the greater tuberosity using an oscillating saw. The tendon (#P9424, Sigma–Aldrich, St. Louis, MO) and a combination and bone chip were wrapped in a silicone tube to prevent of anti-pFAK antibodies (1 ml each, #44-624G, Thermo Fisher spontaneous reattachment. After surgery, sheep were Scientific, Waltham, MA; #sc-11765-R, Santa Cruz Biotech- allowed to move freely in the stable. nology, TX), again under steady rotation at 4˚C. The next

JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2017 COSTAMERE PROTEINS AFTER TENDON TEAR 3

Figure 1. Histologically illustrated temporal changes in tissue composition using representative images of Goldner (left) and oil red O (right) staining at the PRE, 2 W, 16 W, and REP þ 6W time points of the groups TR2 and TR16. On Goldner images, contractile tissue is stained red, the membranes of adipocytes are stained pink, and connective tissue is stained pink and green. On oil red O images, fatty tissue is stained red, while contractile and other types of tissue are unstained. Scale bar denotes 200 mm. day, the protein A-sepharose was washed twice with RIPA the respective membranes: Goat a-mouse antibody #A9917 buffer to remove non-bound proteins. The FAK-pY397 was (Sigma–Aldrich) or mouse a-rabbit antibody #55676 (MP resuspended in 2Â Laemmli buffer, separated from the Biomedicals, OH). The signals were recorded using the PXi immune complex, and stored at À30˚C until detection via System (Syngene, Labgene Scientific SA, Chatel St Denis, Western blot. Switzerland) and quantified using the rectangular mode in Quantity One (Bio-Rad) according to the user manual. To Assessment of Costamere Protein Levels remove inter-membrane variability, signals were first di- Biopsy homogenates were denatured in 2Â Laemmli sample vided by a standard loaded on every gel and after that no buffer and separated on 7.5% SDS–PAGE gels (Bio-Rad statistically significant group differences were detected at Mini-protean TGX stain-free), with 10 mgofproteinloaded baseline, values were scaled per group to set the mean of per lane. To detect FAK-pY397, 10 ml of precipitate was the PRE-values to 1 to display relative changes compared loaded. Proteins were blotted on a nitrocellulose membrane with baseline. using the Bio-Rad Trans-Blot Turbo System (Bio-Rad, Cressier, Switzerland). Equal loading and transfer quality Localization of Costamere Proteins were verified using Ponceau S staining. After blocking in To localize FAK, myosin heavy chain and FAK were co- 5% milk/1% BSA in TBST, the following antibodies were stained on tissue sections following the oil red O staining used to detect the target proteins: FAK-pY397 and total (Fig. 2, right). On another microscopic slide, talin and FAK FAK were detected using a home-made polyclonal rabbit a- were co-stained (Fig. 2, left). In brief, sections were fixed FAK serum (1:1,000) described previously17; gamma- and for 10 min in 4% paraformaldehyde and blocked in a humid meta-vinculin were detected using a mouse a-vinculin chamber in 5% normal goat serum in phosphate buffer serum (gift of Dr. M. A. Glukhova, Paris, France; 1:100); saline and 0.2% triton (PBS-triton) for 1 h at room tempera- and talin and integrin-beta6 were detected using the ture. Primary antibody incubation was performed either in monoclonal mouse a-talin antibody #ab95034 (Abcam, Cam- a mix of mouse a-myosin heavy chain antibodies (a-slow bridge, UK; 1:100) and the polyclonal mouse a-ITGB6 muscle myosin #MAB1628, Millipore Corp., Temecula, CA; antibody #ab169271 (Abcam; 1:500), respectively. Then, the a-fast skeletal myosin #M4276, Sigma–Aldrich; both 1:100) following secondary antibodies (1:20000) were applied on and rabbit anti-FAK #sc-557 (Santa Cruz Biotechnology,

JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2017 4 RUOSS ET AL.

Figure 2. Immunoﬂuorescence images co- stained with talin and FAK (left column) and myosin heavy chain (MyHC) and FAK (right column) followed by consecutive oil red O images (bottom row) of a representative TR16 sheep at 16 W. Scale bar denotes 100 mm.

TX; 1:50), or in rabbit anti-FAK and mouse anti-talin Statistical Analysis (#T3287, Sigma–Aldrich; 1:50). After 1 h, incubation was Data are presented as the mean Æ SD. To detect differences stopped and slides were washed in PBS-triton. Then, between time points and groups, a general linear model with sections were incubated in a mix of the secondary anti- repeated measures was applied with “time point” as the bodies, anti-mouse Alexa Fluor1 488 #A11017 and anti- within-subjects variable (repeated) and “group” as the be- rabbit Alexa Fluor1 555 #A21428 (Thermo Fisher Scien- tween-subjects factor (split-plot ANOVA). A bivariate, two- tific) diluted 1:200 in PBS-triton, for 45 min. After washing tailed Pearson’s test revealed correlations between protein with PBS, nuclei were stained with Hoechst #62249 levels and muscle composition. For the statistical analysis, (Thermo Fisher Scientific; 1:2,000) in PBS for 1 min before SPSS Statistics v22.0 (IBM, Chicago, IL) was used. Statisti- coverslips were mounted. cal significance was defined as p < 0.05.

JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2017 COSTAMERE PROTEINS AFTER TENDON TEAR 5

Figure 3. Muscle volume and composition. Indi- cations above columns refer to alterations in total volume, indications inside columns refer to alterations in the volume of the speciﬁc tissue type. The volume of other types of tissue was calculated from the total volume (contractile tissue þ fatty tissue). Ãp < 0.05, ÃÃÃp < 0.001 for time effect compared with PRE within the same group; þp < 0.05, þþp < 0.01 for time effect compared with 16 W within the same group; #p < 0.05, ### p < 0.001 for group effect compared with TR16 at the same time point. Values are means þ SD.

RESULTS Nandrolone Does Not Influence Total Muscle Volume, but Tendon Release Leads to Reduction of Muscle Volume and Reduces the Loss of Contractile Tissue and Prevents Fat Changes in Tissue Composition Accumulation The initial muscle volume and composition did not In TR16 þ NAN, the total muscle volume was de- differ between groups (Fig. 3). In TR2 and TR16, creased at 16 W (À43.5 Æ 18.3 cm3, p < 0.001 compared tendon release led to a decrease in muscle volume, with PRE). At REP þ 6 W, muscle volume was further which was already detectable at 2 W (À17.0 Æ 5.0 cm3, decreased (À54.7 Æ 19.0 cm3, p < 0.001 compared with p < 0.001 compared with PRE) and was more pro- PRE; À11.2 Æ 5.8 cm3, p ¼ 0.020 compared with 16 W; nounced at 16 W (À35.8 Æ 12.1 cm3, p < 0.001 compared Fig. 3). In the TR16 þ NAN group, the volume of with PRE). At REP þ 6W, muscle volume was further contractile tissue was reduced at 16 W (À43.7 Æ 22.1 decreased (À51.2 Æ 15.0 cm3, p < 0.001 compared with cm3, p < 0.001 compared with PRE) and at REP þ 6W PRE; À15.3 Æ 12.8 cm3, p ¼ 0.004 compared with 16 W; (À64.3 Æ 35.0 cm3, p < 0.001 compared with PRE; Fig. 3). Microscopy images representing the relative À20.5 Æ 18.7 cm3, p ¼ 0.018 compared with 16W). Com- distribution of tissue types are shown in Figure 1 and pared with TR16, the volume of contractile tissue in the percent areas from histological analysis that were TR16 þ NAN was significantly larger at 16 W used to calculate the volumes of contractile, fatty, and (þ20.7 cm3, p ¼ 0.046) and REP þ 6W (þ18.8 cm3, remaining tissue are presented in Figure 4. p ¼ 0.036; Fig. 3). The volume of fatty tissue remained In TR2 and TR16, the volume of contractile tissue unchanged in the nandrolone-treated sheep at both remained unchanged at 2 W (À2.0 Æ 25.6 cm3, p ¼ 0.859 16 W (À4.0 Æ 7.0 cm3, p ¼ 0.368) and REP þ 6W (À4.6 compared with PRE), but was significantly decreased Æ 8.0 cm3, p ¼ 0.283) compared with PRE. This corre- at 16 W (À59.4 Æ 10.2 cm3, p < 0.001 compared with sponds to a significantly lower volume of fatty tissue PRE). Contractile tissue was further reduced at REP compared with TR16 at both 16 W (À27.8 cm3, þ 6W (À77.9 Æ 17.9 cm3, p < 0.001 compared with PRE; p < 0.001) and REP þ 6W (À34.3 cm3, p < 0.001; À18.6 Æ 16.7 cm3, p ¼ 0.028 compared with 16 W; Fig. 3). Nandrolone did not influence the volumes of Fig. 3). The volume of fatty tissue was reduced at 2 W other types of tissue (Fig. 3). (À6.4 Æ 5.9 cm3, p ¼ 0.045 compared with PRE) but then increased at 16W (þ23.6 Æ 12.8 cm3, p < 0.001 Tendon Release Leads to a Relative Deactivation of FAK compared with PRE) in TR2 and TR16, respectively. on the Y397 Site, Which Is Prevented by Nandrolone At REP þ 6W (þ29.4 Æ 11.6 cm3, p < 0.001 compared Administration with PRE), it was similar to 16W (þ5.8 Æ 11.1 cm3, Representative Western blot images are shown in p ¼ 0.186; Fig. 3). Fatty tissue measured in the biopsy Figure 5. The majority of FAK protein is localized sample correlated quantitatively with the fat portion around muscle fibers and not fatty tissue (Fig. 2). Its (r ¼ 0.756, p < 0.001, N ¼ 66) assessed using the Dixon relative activity, expressed as the ratio of Y397- method (described by Gerber et al.)6 on the MRI scans phosphorylated FAK per total FAK, remained un- (data not shown). Tendon release did not influence the changed after 2 weeks of tendon release (À8.2 Æ 41.0%, volume of other tissues (i.e., non-myocellular and non- p ¼ 0.545 compared with PRE), but was significantly fatty tissue; Fig. 3). reduced at 16 W (À27.1 Æ 20.6%, p ¼ 0.039 compared

Figure 4. Relative tissue distribution assessed on biopsy cross-sections. Indications inside columns refer to alterations in the % area of the speciﬁc tissue type. The % of other types of tissue was deﬁned as 100% (contractile tissue þ fatty tissue). Ãp < 0.05, ÃÃÃp < 0.001 for time effect compared with PRE within the same group; ##p < 0.01, ###p < 0.001 for group effect compared with TR16 at the same time point. Values are means þ SD.

JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2017 6 RUOSS ET AL.

with PRE; Fig. 7A). Instead, the 190 kDa fragment of talin was increased already at 16 W (þ122.4 Æ 60.3%, p ¼ 0.003) and remained elevated at REP þ 6W (þ161.1 Æ 84.4%, p ¼ 0.026 compared with PRE; Fig. 7B). Correlation analysis revealed that the abun- dance of the 190 kDa fragment of talin is tightly associated with the levels of FAK (r ¼ 0.801, p < 0.001; Fig. 8). Cleavage of talin was also associated with the loss of contractile tissue (r ¼À0.407, p < 0.001; Fig. 8). Similar to full length talin, gamma-vinculin was not Figure 5. Representative Western blot images of focal adhesion affected by tendon release, but was increased with kinase (FAK), its phosphorylated form FAK-pY397, full length talin and its 190 kDa fragment, gamma- and meta-vinculin, and subsequent repair (þ105.9 Æ 111.6%, p ¼ 0.017 com- integrin-beta6. Relative protein amounts were assessed at base- pared with PRE; Fig. 7C). Meta-vinculin remained line (PRE), 2 weeks (2 W), or 16 weeks (16 W) after tendon release and 6 weeks after repair (REP þ 6 W). The measurements unaffected by tendon release and repair (Fig. 7D). The in the contralateral control (CC) took place at the end of the integrin-beta6 protein level dropped after 2 weeks of intervention. tendon release (À44.6 Æ 36.0%, p ¼ 0.029) and the resting level was reestablished at 16 W. Repair did not with PRE). At REP þ 6 W, the initial level of relative influence integrin-beta6 protein expression (Fig. 7E). FAK activity was not reestablished (À34.2 Æ 19.5%, p ¼ 0.010 compared with PRE; Fig. 6C). The total amount of FAK protein was unchanged at 2 W but Nandrolone Prevents Changes in Costamere Turnover tended to increase after 16 weeks of tendon release After Tendon Release (þ44.6 Æ 33.5%, p ¼ 0.062 compared with PRE), and In the TR16 þ NAN group, tendon release had no effect was only significantly increased after repair compared on the relative quantities of talin, its 190 kDa frag- with baseline (þ94.6 Æ 61.5%, p ¼ 0.012; Fig. 6A). Nan- ment, gamma- and meta-vinculin, and integrin-beta6 drolone prevented the deactivation of FAK on the (Fig. 7A–E). Instead, 6 weeks after repair, the amount Y397 site, after 16 weeks of tendon release, and after of full length talin protein was significantly decreased the subsequent 6 weeks of repair (Fig. 6C), as the total compared with TR16 (À65.0%, p ¼ 0.031; Fig. 7A). amount of FAK (Fig. 6A) remained unchanged. Concomitantly, the 190 kDa fragment of talin was increased at REP þ 6 W compared with baseline Tendon Release and Subsequent Repair Leads to Increased (þ175.7 Æ 195.9%, p ¼ 0.017; Fig. 7B). Compared with Costamere Turnover TR16, nandrolone kept the gamma-vinculin protein Representative Western blot images are shown in level similar to baseline after repair (Fig. 7C). Further- Figure 5. Tendon release did not exert an effect on more, after repair, meta-vinculin and integrin-beta6 talin quantity, but 6 weeks of repair significantly quantities were not distinguishable from baseline or increased it (þ241.4 Æ 160.4%, p < 0.001 compared the respective level in the TR16 group (Fig. 7D and E).

Figure 6. Relative protein quantities of (A) total FAK, (B) FAK-pY397, and (C) the amount of FAK- pY397 per total FAK. Ãp < 0.05, ÃÃp < 0.01 for time effect compared with PRE within the same group. Values are means þ SD.

JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2017 COSTAMERE PROTEINS AFTER TENDON TEAR 7

DISCUSSION of fat but not contractile tissue (Fig. 3). Liu et al.27 The purpose of this study was to define the RC muscle showed previously, in a rat model, that fat accumula- composition after tendon release in sheep and to tion is not detectable at 2 weeks after RC tendon describe costamere turnover with and without weekly release; while, it is present after 6 weeks. Also in doses of nandrolone. In this model, fat accumulated sheep, fat accumulation was detectable on MRI scans and costamere turnover increased at 16 weeks, but not after 6 weeks,6 indicating the turning point for fat 2 weeks after tendon release and these effects were accumulation is between 2 and 6 weeks after tendon prevented by nandrolone. Furthermore, nandrolone release. As blood flow, and thereby delivery of mitigated the loss of contractile tissue. substrates, is contraction dependent,28 we hypothe- Assessments of total muscle volumes via MRI size that the unloaded muscle has to live on its local revealed that muscle remodeling starts immediately energy stores (i.e., intramuscular fatty tissue), lead- after tendon release, as the total muscle volume was ing to decreased fat content until pathological fat already reduced at 2 weeks (Fig. 3). This loss in accumulation occurs. The findings that the contrac- volume continued until 16 weeks and was not tile tissue volume remained unchanged after 2 weeks stopped by repair (Fig. 3). The relative loss of muscle and was significantly decreased after 16 weeks volume corresponds well to the magnitude and time (Fig. 3) support the conclusion that pathological course of atrophy observed in humans during bed muscle loss does not start before 2 weeks after the rest, unilateral limb suspension, immobilization, and injury. spaceflight studies,26 indicating that the ovine model The present study revealed that nandrolone pro- mightalsobesuitableforreplicationofhuman tects contractile tissue. The mitigated loss of contrac- muscle unloading situations other than the RC tear. tile tissue is the reason for similar total muscle Interestingly, the reduction in muscle volume after volumes with/without nandrolone despite significantly 2 weeks is, in part, explained by a decreased volume lower fat content in the nandrolone-treated sheep

Figure 7. Relative quantities of the costamere proteins. (A) Full length talin, (B) the proteolytic 190 kDa fragment of talin, (C) gamma-vinculin, (D) meta-vinculin, and (E) integrin-beta6. Ãp < 0.05, ÃÃp < 0.01, ÃÃÃp < 0.001 for time effect compared with PRE within the same group; þp < 0.05, þþp < 0.01 for time effect compared with 16 W within the same group; #p < 0.05 for group effect compared with TR16 at the same time point. Values are means þ SD.

JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2017 8 RUOSS ET AL.

Figure 8. Pearson correlations for all groups (TR2, TR16, TR16 þ NAN) and all time points (PRE, 2 W, 16 W, REP þ 6 W, CC). Results are presented as the correlation coefﬁcient r and p-value; N ¼ 66.

(Fig. 3). It was shown previously that nandrolone distribution (compare Fig. 3 with Fig. 4). Conversely, reduces unloading-induced loss of muscle protein the assessment of total muscle volume alone does not content in mice.19 The present study confirmed this allow conclusions to be drawn about its composition also occurs with RC tendon release in sheep. Further- (Fig. 3). more, exclusive assessment of the relative tissue The breakdown of contractile tissue has to be distribution does not sufficiently describe shifts in coupled to the disassembly of its structural tissue types if the total volume, which the distribution anchors.10 The costamere is such an anchor and the is related to, varies throughout the intervention, for mechanosensor, FAK, its main regulator, is very example, contractile tissue in the TR16 þ NAN group sensible to alterations of muscle loading.15 The at 16 W was lost without a relative change in tissue relative decrease of FAK-pY397 (Fig. 6) reflects

JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2017 COSTAMERE PROTEINS AFTER TENDON TEAR 9 reduced loading, which was previously shown after CONCLUSIONS unloading of human m. vastus lateralis.16 Similar to In the ovine model of RC tears, muscle atrophy starts our results, total FAK protein tended to increase in with the tendon tear and takes place in two stages. In a human model with unloading by limb suspension16 the first 2 weeks, atrophy is associated with reduced and bedrest.29 Apart from FAK, one of the main fat content and preservation of contractile tissue. structural costamere components, the protein talin, Thereafter, there is substantial fat accumulation and was also affected by tendon release. The increase of loss of contractile tissue. Degradation of contractile the 190 kDa talin fragment at 16 W (Fig. 7) may be tissue is tightly associated with increased costamere indicative of increased costamere turnover, as the turnover and can be mitigated by the administration rise in the fragment did not decrease the amount of of nandrolone decanoate. full length protein; thus, the full length talin seems to be replaced immediately. This 190 kDa fragment OUTLOOK of talin originates from cleavage of the full length Future studies should address whether the repair of protein by the Ca2þ-activated protease calpain.30 The acute tears is possible and successful if started within calpain system is responsible for initial cleavage of 2 weeks after the tear, as contractile tissue is not structural proteins like the costamere to release quantitatively lost by this time point, at least in this sarcomeres and make them accessible for quantita- animal model. The finding that intramuscular fat tive degradation by the proteasome system.10 Indeed, decreases before it accumulates leads to questions the increased cleavage of talin was associated with about the timing of this turning point and what causes loss of contractile tissue (Fig. 8). There were no it. Moreover, the functional relevance of costamere changes in costamere turnover (Figs. 6C and 7A and protein cleavage on the degradation of contractile B) or loss of contractile tissue (Fig. 3) at 2 weeks; tissue needs to be determined. In addition, the clinical while, both were present at 16 weeks after tendon benefit of early administration of nandrolone dec- release. Nandrolone treatment prevented both the anoate needs to be further elaborated. decrease of relative FAK activation and talin cleavage by calpain, probably by lowering Ca2þ-levels19 AUTHORS’ CONTRIBUTIONS and thereby decreasing calpain activity.10,12 In gen- Conception and design of research: MF, CG; performed eral, costamere protein levels seem to be predictive animal experiments: KW, DCM, BVR, MCB; performed of contractile tissue volume (and vice versa; Fig. 8), muscle analysis: SR, CBM; analyzed data: SR, CBM, but further study is needed to determine whether KW; interpreted the results of experiments: SR, MF, there is a causal connection between preservation of CG; funding: MF, CG; prepared figures: SR; drafted the normal costamere turnover and mitigated loss of manuscript: SR; edited and revised the manuscript: SR, contractile tissue. Interestingly, repair increased MF, CG, BVR; and approved the final version of the the relative amounts of FAK, talin, and gamma- manuscript: All authors. vinculin in the TR16 group, indicating that the number of costameres increased, as talin and gamma-vinculin are some of the main structural ACKNOWLEDGMENTS 11 We thank Dr. Marina A. Glukhova for providing the a- components of the costamere and FAK acts as a 17 vinculin antibody. Furthermore, we thank MSc Celine Ferrie main regulator. for performing the Goldner and oil red O staining and This study has several limitations. First, the muscle providing technical assistance. Also, we acknowledge the biopsy was harvested from one site only and does not effort of Dr. Karina Klein, the anesthesia team, and Eric necessarily represent the entire muscle; although, Parigoris. This work has been supported by RESORTHO and fatty tissue measured in the biopsy correlated quanti- the Swiss National Science Foundation (grant #149786), tatively with the fat portion measured on MRI scans. Switzerland. Furthermore, the sample was harvested from the distal third of the lateral portion of the muscle, which REFERENCES is close to the myotendinous junction. This area was 1. Sher JS, Uribe JW, Posada A, et al. 1995. Abnormal findings previously described as having the highest degree of on magnetic resonance images of asymptomatic shoulders. 31 32,33 J Bone Joint Surg Am 77:10–15. fat accumulation in rat and rabbit models of RC 2. Yamaguchi K, Ditsios K, Middleton WD, et al. 2006. The tears. Due to potential regional differences, the calcu- demographic and morphological features of rotator cuff lated volumes of contractile, fatty, and other types of disease. A comparison of asymptomatic and symptomatic tissue should be considered as approximate values. shoulders. 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